Highly permissive cell lines for hepatitis c virus replication

ABSTRACT

This invention relates generally to cells and cell lines that are permissive for hepatitis C virus (HCV) replication, and methods and materials for making and using them.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support under grant numbersCA57973 and AI40034. The U.S. Government may have certain rights in thisinvention.

BACKGROUND

1. Field of the Invention

This invention relates generally to cells that are permissive forhepatitis C virus (HCV) replication, and methods and materials formaking and using them.

2. Related Art

Adaptive mutations in the HCV non-structural proteins that increase RNAreplication, and the frequency of Huh-7 cells supporting detectablelevels of replication, have been identified previously. (Blight, K. J.et al, Science 290:1972-1974, 2000; Guo, J. T. et al., J. Virol.75:8516-8523, 2001; Kreiger, N. et al., J. Virol. 75:4614-4624, 2001;Lohmann, V. et al., J. Virol. 75:1437-1449, 2001). In particular,replacement of the Serine residue with Isoleucine at position 2204 inNS5A permits HCV replication in ˜10% of transfected Huh-7 cells (a20,000-fold improvement over non-mutated HCV) and increases replicationto a level sufficient for the detection of HCV RNA early aftertransfection. (Blight et al., 2000). The low frequency of Huh-7 cellssupporting HCV replication suggests that the cellular environment may bea major determinant of HCV replication efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematic representations of various HCV RNAs.

FIG. 2 shows selection of cell lines highly permissive for HCVreplication.

FIG. 3 shows detection of HCV proteins and RNA in Huh-7.5 and Huh-7cells transiently transfected with HCV RNA.

FIG. 4 shows HCV RNA accumulation after transfection of Huh-7.5 cellswith full-length HCV RNA.

FIG. 5 shows effects of alternative substitutions at position 2204 onHCV RNA replication.

FIG. 6 shows effects of combining NS5A adaptive mutations on HCV Rnareplication.

FIG. 7 shows effects of combining NS3 and NS5A mutations on HCVreplication.

FIG. 8 shows effect of S2194A and S2194D mutations on HCV RNAreplication.

DESCRIPTION

As used herein the term “permissive” for HCV replication, in referenceto a particular cell or cell line, means that the particular cell orcell line supports HCV replication at a frequency that is greater thanthat of the cell or cell line from which it was derived. For example, incertain embodiments described herein, Huh-7 sublines support a higherfrequency of HCV replication than the Huh-7 cell line from which theywere derived. Thus, the sublines are said to be permissive for HCVreplication. In some embodiments, the cell or cell line supportsreplication of HCV at a frequency of between about 10% to about 30%. Inother embodiments, the cell or cell line supports replication of HCV ata frequency of between about 10% and about 75%.

The term “cured” refers to cells substantially free of self-replicatingHCV RNA. Example 1 provides a description of one means for curing cells,within the meaning of that term as used herein.

The term “transfection” refers to the infection of a cell with apolynucleotide. The polynucleotide may be either DNA or RNA. Methods oftransfecting a cell are known in the art, any of which may be used.

Frequency is ascertained by determining the percent of cells havingreplicating HCV RNA. One easy way to measure frequency is to determinethe percentage of cells that exhibit a characteristic conferred by theHCV RNA, and any method known in the art for accomplishing this issuitable. The examples herein describe such a method utilizing HCV RNAcomprising a neo gene and G418 selection.

Unless otherwise noted, conventional techniques of cell culture,molecular biology, microbiology, recombinant DNA and immunology areemployed, all of which are within the skill of the art and are describedin the literature.

In order to obtain cell lines permissive for HCV replication, clonal andpopulation Huh-7 cell lines supporting adapted and non-adaptedsubgenomic or full-length RNA replication were cured of HCV RNA bytreatment with interferon (IN). Since HCV replication can be readilyblocked by IFN, prolonged treatment with IFN cures cells of HCV RNA. Ahigher percentage of cured cells were able to support HCV replicationand facilitated the detection of both subgenomic and full-lengthreplication by multiple assays. Thus, one embodiment described hereincomprises a method of making cells and cell lines that are permissivefor HCV replication. Such a method comprises curing HCV infected cells.The cured cells may then be assayed to determine the frequency at whichcells support HCV replication.

The cells may be any vertebrate cells that are capable of supportingadapted or non-adapted HCV RNA replication. The methods described hereinfor making cells and cell lines are believed to be applicable to anycell type that is capable of supporting adapted or non-adapted HCV RNAreplication. Such cells may include, for example, hepatocytes, T-cells,B-cells, or foreskin fibroblasts, and may be mammalian or morespecifically human cells. A particularly useful cell type is hepatocytecells. For example, Huh-7 cells have been shown to support HCV RNAreplication.

It is demonstrated herein that Huh-7 sublines that have been cured arepermissive for HCV RNA replication. For the replicon containing thehighly adaptive NS5A S2204I mutation, at least 30% of the Huh-7.5 cellscan be transduced to G418 resistance. A comparable fraction was positivefor the NS3 antigen by FACS. Similarly, for the SG replicons lacking neo(FIG. 1), at least 50% initiation efficiency was achieved. Data frommore sensitive FACS analysis (data not shown) indicates that >75% of thecells that survive the transfection procedure harbor replicating HCVRNAs. These permissive cells were obtained by curing replicon-containingcell clones with IFN.

Other embodiments comprise methods of making cells and cell lines thatare permissive for HCV replication. Such methods comprise curinginfected cells, and subsequently assaying sublines to determine thefrequency with which a particular subline supports HCV replication.Sublines that are particularly permissive may then be identified. Theinfected cells may be cured by any means. For example, treatment withinterferon-α is an effective means of curing cells of HCV infection,although any effective means of curing may be utilized within the scopeof this invention.

The most highly permissive sublines (Huh-7.5 and Huh-7.8) were obtainedfrom G418-selected clones that harbored replicons without adaptivechanges in the NS3-5B region (at the population sequence level). Thesecells may represent a subpopulation of the original Huh-7 parental linethat are permissive for replication of unmodified replicons as well asmore permissive for replicons with adaptive mutations. Curing of otherreplicon-containing cell lines did not always yield a cell populationthat was more permissive for the replicons tested. For instance, curingthe Huh-7 population containing the SG-Neo (S2204I) replicon, yielded acell substrate that was unchanged in its ability to support SG-Neo(S2204I), but less efficient (23-fold) for initiation of SG-Neo (5AΔ47)(FIG. 2).

The ability to study HCV replication directly after transfection,without the need for G418 selection, allowed for examination ofreplication of subgenomic replicons lacking neo as well as full-lengthHCV genome RNAs. Initial attempts to create a monocistronic replicon byfusing cellular ubiquitin in-frame between the first 12 amino acids ofcore and NS3 {SG-5′Ub-NS3 (S2204I); FIG. 1} were unsuccessful. Abicistronic derivative with ubiquitin fused to NS3-5B was viable,suggesting that the failure of SG-5′Ub-NS3 (S2204I) to replicate was notdue to a defect in processing at the ubiquitin/NS3 junction (data notshown). Rather, the fusion of the ubiquitin-coding sequence near the HCV5′ NTR may have interfered with translation due to the formation ofdeleterious RNA secondary structures or RNA replication, by disruptingRNA elements that lie in the HCV 5′ NTR or its complement. Fusion of theHCV 5′ NTR to the EMCV IRES yielded a subgenomic replicon thatreplicated better than SG-Neo (S2204I) (FIG. 3). Why deletion of thefirst cistron (sequences encoding C-Neo fusion) from the bicistronicSG-Neo (S2204I) stimulated replication is unknown, but may result fromenhanced translation of the replicase due to abrogated binding of the40S ribosomal subunit to the usual HCV translation initiation site anddiminished competition between the EMCV and HCV IRES elements (J.Marcotrigiano and C. M. Rice, unpublished results).

A similar picture was observed for replication of the full-lengthconstructs containing the NS5A S2204I adaptive change (FIG. 4). InHuh-7.5 cells, the bicistronic construct containing the C-Neo cistron{FL-Neo (S2204I); FIG. 1} initiated replication less efficiently thanthe RNA with the HCV 5′ NTR fused to the EMCV IRES {FL-5′HE (S2204I);FIG. 1}. The FL construct with the unmodified HCV genome (except for theS2204I substitution in NS5A) was better at initiating replication thanRNA where translation was mediated by the EMCV IRES (FIG. 4),demonstrating that EMCV IRES-driven translation is not required for HCVreplication in Huh-7.5 cells, thus allowing the study of unmodified HCVgenomic RNAs. This latter point could certainly impact the ability ofHCV RNAs to be packaged into infectious particles. However, in our hands(K. J. Blight and C. M. Rice, unpublished results) and in a recentreport (Pietschmann, T. et al., J. Virol. 76:4008-4021, 2002) selectivepackaging of these unmodified FL RNAs was not observed in Huh-7 cells.Full-length HCV RNAs were less efficient at establishing replicationthan the corresponding adapted subgenomic replicons, suggesting thataddition of the structural-NS2 coding region inhibits HCV replicationinitiation. Whether this is due to the encoded proteins or RNA elementsthat lie in this region (or both) (31) is currently unclear. We haveobserved that the levels of HCV replication (as measured by HCV proteinand RNA levels) of S2204I-containing FL and FL-Neo RNAs, but notsubgenomic replicons, is dependent upon the cell cycle which mightcompromise the ability of the full-length RNAs to initiate replicationin an unsynchronized transfected cell population (Balfe et al.,submitted).

In an attempt to further enhance HCV replication in cell culture, theeffect of other amino acid substitutions at position 2204 in NS5A wasexamined. Efficient subgenomic RNA replication was observed for Ile andVal and to a much lesser extent, Ala at position 2204 (FIG. 5). Val orIle are small, β-branched, non-polar residues, whereas Ala has similarproperties, but is not β-branched. In contrast, polar residues such asTyr, Glu, Thr, Ser or Asp at position 2204 severely impaired HCVreplication (FIG. 5), suggesting that replication favors non-polarresidues at this locus. It is interesting to note that Ser is foundnaturally at position 2204 for this genotype 1b isolate (Lohmann, V. etal., Science 285:110-113, 1999) and is conserved between other HCVgenotypes (Tanji, Y. et al., J. Virol. 69:3980-3986, 1995), suggestingthat this residue may be important for HCV replication and/orpathogenesis in vivo.

Combining NS5A adaptive mutations resulted in replicons that were eitherimpaired (A2199T+S2204I) or unable to replicate (S2197P+A2199T+S2204I)in Huh-7.5 cells (FIG. 6). Incompatability of adaptive mutationselsewhere in the HCV NS coding region has been previously described(Lohmann, V. et al., J. Virol. 75:1437-1449). For example, combining anadaptive mutation in NSSB (R2884G) with either NS4B (P1936S) or NS5A(E2163G) drastically reduced the efficiency of G418-resistant colonyformation. On the other hand, combining certain NS3 and NS5A adaptivemutations can increase replication efficiency (Krieger, N. et al., J.Virol. 75:4614-4624, 2001). However, despite the observation thatmutations E1202G and T1280I in NS3 act synergistically with S2197P inNS5A to increase the replication efficiency (Krieger et al., 2001, andK. J. Blight and C. M. Rice, unpublished results), engineering these NS3changes into SG-5′HE (S2204I) did not enhance replication in our system(FIG. 7). These results again underscore the empirical nature ofoptimizing adaptive mutations with different Huh-7 cellularenvironments.

The phosphorylation of NS5A is conserved among divergent HCV genotypes(Reed, K. E. et al, J. Virol. 71:7187-7197, 1997) suggesting that itplays an important role in the virus life cycle. We previously showedthat NS5A hyperphosphorylation is not essential for HCV replication(Blight, K. J. et al., Science 290:1972-1974, 2000). Following therecent identification of S2194 as a major phosphate acceptor site forsubtype 1b (Katze, M. G. et al, Virology 278:501-513), Ala or Asp wassubstituted at this position and the effect on HCV replication wasexamined in the context of the S2204I adaptive mutation. Given theincompatibilities observed when combining NS5A mutations, the absolutereplication efficiencies of the different mutants could not beevaluated, however replicating RNAs were recovered that harbored thesesubstitutions at the 2194 locus. These results show that phosphorylationat S2194 is not an absolute requirement for replication of this subtype1b isolate.

Various embodiments of the invention are described in the followingexamples. These examples are to be considered exemplary only, and arenot intended to be limiting.

EXAMPLES Example 1 Cell Culture and Interferon Treatment

Huh-7 cell monolayers were propagated in Dulbecco's modified minimalessential medium (DMEM) supplemented with 10% heat-inactivated fetalbovine serum (FBS) and 0.1 mM non-essential amino acids (DMEM-10% FBS).For cells supporting subgenomic replicons, 750 μg/ml G418 (Geneticin;Gibco-BRL) was added to the culture medium. Replicon-containing Huh-7cells were cured of HCV RNA by initially passaging cells twice in theabsence of G418. On the third passage cells were cultured with 100 IU/mlof human leukocyte-derived IFN (Sigma-Aldrich). After 3-4 days,confluent monolayers were trypsinized, plated and cultured for 24 hbefore the addition of IFN. Cells were passaged a total of four times inthe presence of IFN and prior to the fourth passage cells were grown for3 days without IFN. Cured cell lines were expanded and cryopreserved atearly passage levels. Further experiments were conducted using cellsthat been passaged less that 20-30 times from these cryopreserved seedlots.

Example 2 Plasmid Construction

Standard recombinant DNA technology was used to construct and purify allplasmids. Primed DNA synthesis was performed with KlenTaqLA DNApolymerase (kindly provided by Wayne Barnes, Washington University, St.Louis), and regions amplified by PCR were confirmed by automatednucleotide sequencing. Plasmid DNAs for in vitro transcription wereprepared from large-scale bacterial cultures and purified bycentrifugation in CsCl gradients.

All nucleotides (nt) and amino acid numbers refer to the location withinthe genotype 1b Con1 full-length HCV genome (Genbank Accession no.AJ238799) commencing with the core-coding region. This sequence wasassembled from chemically synthesized DNA oligonucleotides in astep-wise PCR assay essentially as described previously (5). Briefly,10-12 gel-purified oligonucleotides (60-80 nt in length) with uniquecomplementary overlaps of 16 nt were used to synthesize cDNAs spanning600-750 bases. The final PCR products were purified, digested withappropriate restriction enzymes, and ligated into the similarly cleavedpGEM3Zf(+) plasmid vector (Promega). Multiple recombinant clones weresequenced, correct clones identified and overlapping cDNA fragmentsassembled into the contiguous genomic sequence:5′NTR-C-E1-E2-p7-NS2-3-4A-4B-5A-5B-3′NTR (pHCVBMFL). The selectablereplicon, pHCVrep1bBartMan/AvaII {SG-Neo (wt); FIG. 1} and thederivatives, pHCVrep1b/BBVII {SG-Neo (S2204I)} and pHCVrep1b/BBI {SG-Neo(5AΔ47)}, containing the NS5A adaptive mutations, S2204I and an in-framedeletion of 47 amino acids (Δ47aa) between nt 6960 and 7102,respectively, have been described (5) (FIG. 1). The plasmidpHCVBMFL/S2204I {FL (S2204I); FIG. 1} contains the full-length genomewith the NS5A adaptive change S2204I. For the genomic and subgenomicconstructs, NS5B polymerase defective derivatives were generatedcarrying a triple amino acid substitution, changing the Gly-Asp-Asp(GDD) motif in the active site to Ala-Ala-Gly (AAG) (5), and throughoutthis report are referred to as pol⁻.

The plasmid pC-Ub-NS3/HCVrepBBVII {SG-5′Ub-NS3 (S2204I); FIG. 1}containing ubiquitin instead of the neo gene and EMCV IRES wasconstructed as follows. An AscI-SacI digested PCR fragment amplifiedfrom pHCVrep12/Neo (Blight et al., unpublished results) with primers1289 and 1290 (Table 1) and the SacI-BsrGI portion of a second PCRproduct generated using the primer pair 1291/1292 (Table 1) withpHCVrep1b/BBVII were ligated between the XbaI and BsrGI sites ofHCVrep1b/BBVII together with the XbaI-AscI fragment from HCVrep1b/BBVII.To delete the neo gene from pHCVrep1b/BBVII, synthetic overlappingoligonucleotides 1287 and 1288 (Table 1) were hybridized and extended tocreate the junction between the 5′ NTR and the EMCV IRES. This productwas digested with ApaLI and AclI, and inserted, together with XbaI-ApaLIand AclI-EcoRI fragments from pHCVrep1b/BBVII, into XbaI-EcoRI digestedpHCVrep1b/BBVII. Thisconstruct was named p5′NTR-EMCV/HCVrepBBVII{SG-5′HE (S2204I); FIG. 1}. To replace S2204I with NS5AΔ47, theEcoRI-XhoI fragment from pHCVrep1b/BBI was ligated into similarlycleaved p5′NTR-EMCV/HCVrepBBVII, generating p5′NTR-EMCV/HCVrepBBI{SG-5′HE (5AΔ47); FIG. 1}.

The plasmid p5′NTR-EMCV/HCVFLBM(S2204I) {FL-5′HE (S2204I); FIG. 1} wascreated by ligating the XbaI-HindIII fragment fromp5′NTR-EMCV/HCVrepBBVII, the HindIII-AatII fragment of a PCR productamplified from p5′NTR-EMCV/HCVrepBBVII using primers 1293 and 1294 andthe AatII-NotI fragment from pHCVBMFL/S2204I into pHCVBMFL/S2204Ipreviously digested with XbaI and NotI. The selectable bicistronicfull-length HCV clone pHCVBMFL(S2204I)/Neo {FL-Neo (S2204I); FIG. 1} wasassembled by ligating the XbaI-HindIII fragment from pHCVrep1b/BBVII andthe HindIII-EcoRI fragment from p5′NTR-EMCV/HCVBMFL(S2204I) between theXbaI and EcoRI sites of pHCVrep1b/BBVII.

To obtain plasmids with mutations at position 2204, and to introducesingle A2199T or double S2197P/A2199T mutations intop5′NTR-EMCV/HCVrepBBVII, PCRs were first performed usingp5′NTR-EMCV/HCVrepBBVII as a template with the reverse primer 1030 andone of the following mutant forward primers: 1319 (S2204V), 1320(S2204A), 1322 (S2204Y), 1324 (S2204E), 1325 (S2204T), 1184 (S2204D),1326 (A2199T+S2204I) and 1327 (S2197P+A2199T+S2204I) (Table 1).PCR-amplified products were digested with BlpI and XhoI and cloned intothese sites in p5′NTR-EMCV/HCVrepBBVII. S2204 was engineered byinsertion of the EcoRI-XhoI fragment from pHCVrep1bBartMan/AvaII intosimilarly cleaved p5′NTR-EMCV/HCVrepBBVII.

To engineer the mutation, Q1112R, into p5′NTR-EMCV/HCVrepBBVII in orderto create p5′NTR-EMCV/HCVrepCloneA (Q1112R+S2204I), nt 3640-3991 of NS3were PCR amplified from p5′NTR-EMCV/HCVrepBBVII using mutant primer 1358and oligonucleotide 885 (Table 1). The resulting product was digestedwith BsrGI and EagI and combined in a ligation reaction mixture with theEagI-EcoRI and BsrGI-EcoRI fragments from p5′NTR-EMCV/HCVrepBBVII. Thedouble mutation (E1202G+T1280I) in NS3 was created via a multistepcloning procedure. First, a PCR fragment amplified fromp5′NTR-EMCV/HCVrepBBVII with forward primer 1359 and reverse primer 1356(Table 1) was digested with ApaLI and XbaI and cloned into EcoRI-XbaIdigested pGEM3Zf(+) together with the EcoRI-ApaLI fragment frompGEM3Zf(+)/HCV1bnt1796-2524, containing nt 3420-4124 in NS3 (K. J.Blight and C. M. Rice, unpublished results), generating the intermediateplasmid pGEM3Zf(+)/HCV1bnt1796-2524NS3*. Second, in a four part cloningstrategy, the BsrGI-BsaAI fragment, excised frompGEM3Zf(+)/HCV1bnt1796-2524NS3*, was inserted, together with fragmentsBsaAI-BssHII and BssHII-EcoRI from p5′NTR-EMCV/HCVrepBBVII, intop5′NTR-EMCV/HCVrepBBVII cleaved with BsrGI and EcoRI. The resultantplasmid was named p5′NTR-EMCV/HCVrepBBVII+NS3* (E1202G+T1280I+S2204I).

The mutations S2194A and S2194D were introduced by using primer pairs5′Ala/1030 and 5′Asp/1030 (Table 1), respectively to PCR amplify nt6897-7186 in NS5A from pHCVrep1b/BBVIII. These mutations wereincorporated into pHCVrep1b/BBVII by replacing the BlpI-XhoI portionwith the corresponding BlpI-XhoI digested PCR product.

Example 3 RNA Transcription

Plasmid DNAs containing full-length and subgenomic HCV sequences werelinearized with ScaI and a poliovirus subgenomic replicon digested withBamHI. The linearized DNAs were phenol:chloroform (1:1) extracted, andprecipitated with ethanol. Pelleted DNAs were washed in 80% ethanol andresuspended in 10 mM Tris-HCl (pH 8.0)/1 mM EDTA (pH 8.0). RNAtranscripts were synthesized at 37° C. for 90 min in a 100 μl reactionmixture containing 40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 12 mM MgCl₂, 2mM spermidine, 10 mM dithiothreitol (DTT), 3 mM of each nucleosidetriphosphate, 0.025 U of inorganic pyrophosphatase (Roche AppliedScience), 100 U of RNasin (Promega), 100 U of T7 RNA polymerase(Epicentre Technologies), and 2 μg of linearized DNA. RNA was extractedwith phenol-chloroform (1:1), ethanol precipitated, and the pelletwashed in 80% ethanol before resuspension in ddH₂O. DNA template wasremoved by three serial DNase digestions for 20 min at 37° C. in 33 mMTris-HCl (pH 7.8), 66 mM KCl, 10 mM MgCl₂ and 5 mM DTT containing 10 Uof DNase I (Roche Applied Science). DNase-digested RNAs were extractedwith phenol:chloroform (1:1), ethanol precipitated and the RNA pelletresuspended in ddH₂0 after washing in 80% ethanol. The RNA concentrationwas determined by measurement of the optical density at 260 nm and theintegrity and concentration confirmed by 1% agarose gel electrophoresisand ethidium bromide staining.

Example 4 Transfection of Cultured Cells

In vitro-transcribed RNA was transfected into Huh-7 and IFN-cured cellsby electroporation. Briefly, subconfluent Huh-7 cells were detached bytrypsin treatment, collected by centrifugation (500×g, 5 min), washedthree times in ice-cold RNase-free phosphate-buffered saline (PBS) andresuspended at 1.25×10⁷ cells/ml in PBS. RNA transcripts (1 μg) weremixed with 0.4 ml of washed Huh-7 cells in a 2-mm gap cuvette (BTX) andimmediately pulsed (0.92 kV, 99 μsec pulse-length, 5 pulses) using a BTXElectroSquarePorator. Pulsed cells were left to recover for 10 min atroom temperature (rt) and then diluted into 10 ml DMEM-10% FBS. Cellswere plated in: (i) 35-mm diameter wells for quantifying HCV RNA and formetabolic labeling experiments; (ii) eight-well chamber slides (BectonDickinson) for immunofluorescence studies or; (iii) 100-mm diameterdishes for fluorescent activated cell sorting (FACS) analysis and G418selection. To determine the efficiency of G418-resistant colonyformation, transfected cells were plated at multiple densities (between1×10³ and 2×10⁵ cells), together with cells transfected with pol RNAtranscripts such that the total cell number was maintained at 2×10⁵cells per 100-mm diameter dish. Forty-eight hours after plating, mediumwas replaced with DMEM-10% FBS supplemented with 1 mg/ml G418. Threeweeks later, G418 resistant foci were fixed with 7% formaldehyde andstained with 1% crystal violet in 50% ethanol to facilitate colonycounting. The G418 transduction efficiency was calculated based on thenumber of G418-selected colonies relative to the number of Huh-7 cellsplated after electroporation.

Transfection efficiency was monitored for each series of RNAs byelectroporating in parallel a poliovirus subgenomic replicon expressinggreen fluorescent protein (GFP; A. A. Kolykhalov and C. M. Rice,unpublished results). Transfected cells were observed for poliovirusreplicon-induced cytopathic effect and GFP expression visualized using afluorescent inverted microscope at 12-16 h posttransfection. After 24 h,the surviving attached cells (presumably not transfected with thepoliovirus replicon) were trypsinized, mixed with trypan blue and viablecells counted to determine the percentage of cells electroporated.

Example 5 Viral RNA Analysis

Total cellular RNA was isolated using TRIZOL reagent (Gibco-BRL)according to the manufacturer's protocol. One-tenth of each RNA samplewas used to quantify HCV-specific RNA levels using an ABI PRISM 7700Sequence Detector (Applied Biosystems). Real time reverse transcription(RT)-PCR amplifications were performed using the TaqMan EZ RT-PCR corereagents (Applied Biosystems) and primers specific for the HCV 5′ NTR:5′-CCTCTAGAGCCATAGTGGTCT-3′ (sense, 50 μM), 5′ CCAAATCTCCAGGCATTGAGC-3′(antisense, 50 μM) and FAM-CACCGGAATTGCCAGGACGACCGG (probe, 10 μM;Applied Biosystems). RT reactions were incubated for 30 min at 60° C.,followed by inactivation of the reverse transcriptase coupled withactivation of Taq polymerase for 7 min at 95° C. Forty cycles of PCRwere performed with cycling conditions of 15 sec at 95° C. and 1 min at60° C. Synthetic HCV RNA standards of known concentration were includedwith each set of reactions and used to calculate a standard curve. Thereal time PCR signals were analyzed using SDS v1.6.3 software (AppliedBiosystems).

Example 6 FACS Analysis

Transfected cell monolayers were removed from 100-mm diameter culturedishes by versene/EDTA treatment and a single cell suspension preparedby passing cells through a 16-gauge needle and a 74 μm pore membrane.Cells were resuspended at 2×10⁶ per ml, an equal volume of 4%paraformaldehyde added to the cell suspensions and incubated for 20 minat rt. Fixed cells were washed twice with PBS and the resultant cellpellet resuspended at 2×10⁶ cells per ml in 0.1% saponin/PBS. Afterincubation for 20 min at rt, cells were stained (1 h at rt) withHCV-specific monoclonal antibodies (mAbs; core (C750), NS3 (1B6) andNS5B (12B7); all generously provided by Darius Moradpour, University ofFreiburg, Freiburg, Germany) diluted to 10 μg/ml in 3% FBS/0.1%saponin/PBS. Cells were washed three times with 0.1% saponin/PBS andbound mAb detected by incubation for 1 h at rt with anti-mouse IgGconjugated to Alexa 488 (Molecular Probes) diluted 1:1000 in 3% FBS/0.1%saponin/PBS. Stained cells were washed three times with 0.1%saponin/PBS, resuspended in FACSflow buffer (BD Biosciences) andanalyzed immediately using a FACS Calibur (BD Biosciences).

Example 7 Indirect Immunofluorescence

Electroporated Huh-7.5 cells seeded in eight-well chamber slides werewashed with PBS and fixed in 4% paraformaldehyde for 20 min at rt. Cellswere washed twice with PBS, permeabilized by incubation with 0.1%saponin/PBS for 20 min at rt and blocked with 3% goat serum for 20 minat rt. The NS5B mAb (12B7) was diluted to 10 μg/ml in 0.1% saponin/3%goat serum/PBS and incubated for 1 h at rt, followed by three washeswith 0.1% saponin/PBS. Bound mAb were detected by incubating for 1 h atrt with anti-mouse IgG conjugated to Alexa 488 diluted 1:1000 in 0.1%saponin/3% goat serum/PBS. Nuclei were stained for 20 min at rt with 10μg/ml Hoechst 33342 (Sigma-Aldrich) in PBS. Unbound fluorescentconjugate was removed by three washes with 0.1% saponin/PBS, cellsmounted in Vectashield (Vector Laboratories) and viewed with afluorescent microscope (Nikon, Eclipse TE300).

Example 8 Metabolic Labeling of Proteins and Immunoprecipitation

Cell monolayers in 35-mm diameter wells were incubated for 0.5-10 h inmethionine-and-cysteine-deficient MEM containing 1/40^(th) the normalconcentration of methionine, 5% dialyzed FBS and Express ³⁵S-proteinlabeling mix (140 μCi/ml; NEN). Labeled cells were washed once with coldPBS and harvested in 200 μl of sodium dodecyl sulfate (SDS) lysis buffer(0.1 M sodium phosphate buffer (pH 7.0), 1% SDS, 1× complete proteaseinhibitor cocktail (Roche Applied Science), 80 μg phenylmethylsulfonylfluoride (PMSF) per ml) and cellular DNA sheared by repeated passagethrough a 27.5-gauge needle. Equal amounts of protein lysates (50 μl)were heated at 75° C. for 10 min and clarified by centrifugation priorto mixing with 200 μl of TNA (50 mM Tris-HCl (pH 7.5), 150 mM NaCl,0.67% bovine serum albumin, 1 mM EDTA, 0.33% triton X-100, 80 μg of PMSFper ml). One-μl of HCV positive patient serum (JHF) was added, andimmune complexes allowed to form by incubation overnight at 4° C. withrocking. Immune complexes were collected by adding 50 μl of prewashedPansorbin cells (Calbiochem) and incubation for 1-2 h at 4° C. withrocking. Immunoprecipitates were collected by centrifugation and washedthree times in TNAS (TNA containing 0.125% SDS) and once with TNE (50 mMTris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 80 μg of PMSF per ml),solubilized by heating at 80 ° C. for 20 min in protein sample bufferand separated on an SDS-10% polyacrylamide gel. Metabolically labeledproteins were visualized by fluorography.

Example 9 Cell Lines Permissive for HCV Replication

From 22 G418-resistant clones (Blight, K. J. 2000), clones Huh-7.5 andHuh-7.8, harboring SG-Neo subgenomic replicons with no amino acidchanges within the HCV NS region, as well as clone Huh-7.4, containing areplicon with the Ser to Ile change at position 2204 in NS5A, werecured. Uncloned population lines Huh-7/S2204I and Huh-7/5AΔ47 (Blight,K. J. 2000), selected with G418 after transfection of subgenomicreplicons containing either S2204I in NS5A {SG-Neo (S2204I); FIG. 1} orthe 47 amino acid NS5A deletion {SG-Neo (5AΔ47); FIG. 1}, were alsotreated with IFN. To exclude the possibility that IFN treatment alonemay alter the ability of Huh-7 cells to support HCV replication, theparental Huh-7 cells were treated with IFN in parallel. Following IFNtreatment, cells were shown to lack HCV RNA by a nested RT-PCR specificfor the 3′ NTR where the detection limit was −10 molecules of HCV RNA(Kolykhalov, A. A., J. Virol. 70:3363-3371) and by sensitivity to G418.

To examine the ability of IFN-cured cell lines to support HCVreplication, three G418-selectable replicons, SG-Neo (S2204I), SG-Neo(5AΔ47) and SG-Neo (wt) (FIG. 1), with G418-transduction efficiencies inparental Huh-7 cells of 10%, 0.2% and 0.0005%, respectively, were used(Blight, K. J. 2000). In vitro-synthesized RNA was electroporated intoIFN-cured cells and after 48 h, G418 selection was imposed and theresulting colonies counted after fixing and staining. The transductionefficiencies were calculated on the basis of the number of G418-selectedcolonies relative to the number of Huh-7 cells plated afterelectroporation. The frequency of Huh-7.5 cells able to support SG-Neo(S2204I) replication was ˜3-fold higher than the parental Huh-7 cells(FIG. 2). For cell lines Huh-7.5 and Huh-7.8, the nunber ofG418-resistant colonies obtained after transfection of SG-Neo (5AΔ47)was significantly higher than for the parental Huh-7 cells (˜33- and9-fold increases, respectively; FIG. 2). The same was true for Huh-7.4,although the increased frequency of colony formation was not as great(˜3-fold; FIG. 2). The SG-Neo (wt) replicon also showed an enhancedreplicative capacity in Huh-7.5, Huh-7.8 and Huh-7.4 cells (10-, 2- and2-fold increases, respectively; FIG. 2).

The two cured cell populations, Huh-7/5AΔ47 and Huh-7/S2204I, showedeither comparable or modest increases in G418 transduction efficienciesafter transfection of the adapted replicon RNA originally present withinthe population line (FIG. 2). The frequency of G418-resistant coloniesincreased ˜2.5-fold when Huh-7/5AΔ47 cells were electroporated withSG-Neo (5AΔ47), whereas transfection with SG-Neo (S2204I) resulted in aslight decrease in the G418 transduction efficiency (FIG. 2). However, a23-fold reduction in colony formation was observed after transfection ofHuh-7/S2204I cells with SG-Neo (5AΔ47) (FIG. 2). No significantdifferences in G418-resistant colony formation were noted between theparental Huh-7 cells and IFN-treated Huh-7 cells (data not shown),indicating that the IFN-mediated curing protocol did not stablyinfluence the ability of these cells to support HCV replication.G418-resistant colonies were not observed when the polymerase defectivereplicon RNA, pol⁻, was transfected in parallel (data not shown). Hence,a higher frequency of cells in the cured clonal lines, in particularthose originally able to support replication of RNAs without adaptivemutations (Huh-7.5 and Huh-7.8), are permissive for HCV replication.

Example 10 HCV Replication in Unselected Huh-7.5 and Huh-7 Cells

Since the cured Huh-7.5 line was the most permissive of those tested, weexamined HCV replication in this subline compared with the parentalHuh-7 cells using a number of different methods. We focused on transientassays that would allow an assessment of HCV replication early aftertransfection without the need for G418 selection. Ninety-six hours aftertransfection with SG-Neo (S2204I) and SG-Neo (5AΔ47) RNA (FIG. 1), totalRNA was extracted from Huh-7.5 and IFN-treated Huh-7 cells and the HCVRNA levels quantified by RT-PCR. The replication-defective replicon,pol⁻, was transfected in parallel to allow discrimination between inputRNA and RNA generated by productive replication. As shown in FIG. 3, thelevels of HCV RNA relative to the por control were consistently higherin the transfected Huh-7.5 cells. Transfection with SG-Neo (S2204I) andSG-Neo (5AΔ47) RNAs resulted in 410- and 28-fold increases,respectively, in Huh-7.5 cells (FIG. 3, lanes 4 and 5), compared to only85- and 6-fold in Huh-7 cells (FIG. 3, lanes 10 and 11). Since theseincreases are measured relative to the replication defectivepol-control, they reflect accumulation of newly synthesized RNA versusdegradation of input RNA. In Huh-7.5 and Huh-7 cells, the level ofresidual pol-RNA declined by about 10-fold at each timepoint. In Huh-7.5cells, RNAs was good replicative abilities {like SG-Neo (S2204I)} tendedto accumulate over time such that about a 10-fold increase was observedby 96 h. Those with lower replicative ability {like SG-Neo (5AΔ47)}remained constant or declined slightly, but never to the level of thepol-control. For Huh-7 cells, the picture was somewhat different. Forexample, SG-Neo (S2204I) RNA remained relatively constant whereas SG-Neo(5AΔ47) RNA decreased over time, but again, not to the extent of thepol− control.

This finding was mirrored by the frequency of NS3-positive cellsmeasured by FACS analysis. The percentage of NS3-positive cells wasconsistently higher in Huh-7.5 cells {21% for SG-Neo (S2204I) and 5% forSG-Neo (5AΔ47); FIG. 3, lanes 4 and 5} compared to Huh-7 cells {3% forSG-Neo (S2204I) and undetectable for SG-Neo (5AΔ47) and pol⁻ RNAs; FIG.3, lanes 7, 10 and 11}. These results confirm our earlier conclusionthat a larger fraction of Huh-7.5 cells support detectable levels of HCVreplication. The lower frequency of HCV antigen-positive cellsquantified by FACS compared to the G418 transduction efficiency isattributable to the sensitivity of FACS analysis, that varies withdifferent HCV-specific antibodies (unpublished observations).

HCV protein accumulation was examined by metabolically labeling cells 96h after transfection. Cell monolayers were labeled with ³⁵S-methionineand -cysteine for 10 h, followed by SDS-mediated lysis andimmunoprecipitation of HCV proteins with a HCV-positive patient serum(JHF) recognizing NS3, NS4B and NS5A (Grakoui, A. et al., J. Virol.67:1385-1395). After separation of labeled proteins by SDS-PAGE, NS3,NS4B and NS5A were only visible in Huh-7.5 cells transfected with SG-Neo(S2204I) (FIG. 3, lane 4). HCV proteins were never detected in Huh-7.5and Huh-7 cells transfected with SG-Neo (5AΔ47), pol⁻ or SG-Neo (S2204I)RNA electroporated Huh-7 cells (FIG. 3, lanes 1, 5, 7, 10 and 11).Similar results were obtained after metabolic labeling of HCV RNA in thepresence of actinomycin D (data not shown). These analyses demonstratethe advantages of using Huh-7.5 cells for rapid analysis of HCVreplication by RNA accumulation, FACS analysis and metabolic labeling ofviral proteins.

Example 11 Replicative Efficiencies of Subgenomic and Genomic HCV RNAs

The ability to monitor HCV replication without selection eliminated theneed for bicistronic replicons and allowed constructs with minimalheterologous elements to be tested. A subgenomic replicon was engineeredin which the HCV 5′ NTR and 12 amino acids of core were fused toubiquitin followed by the NS3-5B coding region (including the S2204Iadaptive mutation in NS5A) and the 3′ NTR {SG-5′Ub-NS3 (S2204I); FIG.1}. In this polyprotein, cellular ubiquitin carboxyl-terminal hydrolasewill cleave at the ubiquitin/NS3 junction to produce NS3 with anauthentic N-terminal Ala residue (2, 21). In vitro-synthesized RNA waselectroporated into Huh-7.5 and Huh-7 cells and the level of HCV RNAquantified 96 h later by RT-PCR. Surprisingly, HCV RNA levels did notdiffer from the por control (data not shown), indicating thatSG-5′Ub-NS3 (S2204I) RNA failed to replicate. It is possible thatubiquitin may interfere with the production of a functional NS3 protein.However, a bicistronic derivative, where expression of ubiquitin/NS3-5Bwas under the control of the EMCV IRES, replicated as efficiently asSG-Neo (S2204I) RNA (data not shown), suggesting that HCV IRES driventranslation may be sensitive to RNA elements present within thecore-ubiquitin coding sequence.

A replicon lacking neo but retaining the EMCV IRES (SG-5′HE derivatives;FIG. 1) was also tested. SG-5′HE (S2204I) and SG-5′HE (5AΔ47) RNA levelsrelative to the pol⁻ control were measured 96 h after transfection andfound to be higher than the selectable versions in both Huh-7.5 andHuh-7 cells (FIG. 3). Moreover, the levels of SG-5′HE (S2204I) andSG-5′HE (5AΔ47) RNA were significantly higher in Huh-7.5 compared toHuh-7 cells (FIG. 3, lanes 2, 3, 8 and 9). Approximately 52% of Huh-7.5cells stained positive for the NS3 antigen after transfection of SG-5′HE(S2204I) RNA, compared to 21% for SG-Neo (S2204I) RNA (FIG. 3, lanes 2and 4). Similarly, a higher frequency of Huh-7.5 cells expressed NS3after electroporation with SG-5′HE (5AΔ47) compared to SG-Neo (5AΔ47)(11% versus 5%; FIG. 3, lanes 3 and 5). As expected, lower frequenciesof NS3-positive cells were observed for transfected Huh-7 cells (FIG.3). The relative amounts of immunoprecipitated ³⁵S-labeled HCV proteinsfrom Huh-7.5 and Huh-7 cells paralleled both the frequency ofNS3-positive cells and relative HCV RNA levels (FIG. 3). These datademonstrate that replicons lacking the neo gene initiate RNA replicationmore efficiently. These constructs, together with the highly permissiveHuh-7.5 subline, are valuable tools for genetic studies on HCV RNAreplication, some of which are described later in this report. Theability of Huh-7.5 cells to support replication of full-length HCV RNAcontaining S2204I in NS5A {FL (S2204I); FIG. 1} was also assessed.Ninety-six hours after transfection of Huh-7.5 and Huh-7 cells, therelative levels of HCV RNA and protein were measured as described above.A 50-fold increase in HCV RNA relative to pol⁻ was observed aftertransfection of Huh-7.5 cells, compared to only a 3-fold increase inHuh-7 cells (FIG. 3, lanes 6 and 12). Similarly, FACS analysis andimmunoprecipitation of metabolically labeled proteins failed to detectHCV antigen expression in FL RNA transfected Huh-7 cells, whereas 14%and 10% of Huh-7.5 cells expressed core and NS3 antigens, respectively,and ³⁵S-labeled NS3 was detectable (FIG. 3, lanes 6 and 12). Thefrequency of core-antigen positive cells was consistently higher thanthat seen for NS3, possibly reflecting differences in antibody affinity.The ability of full-length HCV RNA to establish replication in Huh-7.5cells demonstrates that replication is not dependent upon EMCVIRES-driven translation of HCV-encoded replicase components. In fact,inclusion of the EMCV IRES downstream of the HCV 5′ NTR {FL-5′HE(S2204I); FIG. 1} or creation of a biscistronic construct with the neogene added {FL-Neo (S2204I); FIG. 1} impaired replication relative to FL(2204I) RNA (FIG. 4). It is interesting to note that all of theconstructs containing the complete HCV coding sequence(S2204I-containing FL, FL-5′HE and FL-Neo) were less efficient atestablishing replication compared to subgenomic replicons lacking thestructural-NS2 coding region {eg. SG-5′HE (S2204I); FIG. 4}. Thissuggests that cis RNA elements or proteins encoded in this region of thegenome may downregulate the efficiency of HCV replication in thissystem. Nonetheless, the ability of Huh-7.5 cells to support replicationof both FL and FL-Neo RNAs provides systems that may be useful forstudying steps in particle assembly and examining the impact of theentire HCV protein complement on host cell biology.

Example 12 Effect(s) of Mutations in NS3 and NS5A on HCV RNA Replication

Thus far, the best single mutation that has been identified is theS2204I substitution in NS5A. To examine the importance of Ile at thisposition and to see if replication efficiency could be improved further,a number of other amino acids were tested at this position and comparedthe replication efficiency of these replicons to SG-5′HE (S2204I) or theunmodified parent, SG-5′HE (S2204) (FIG. 5). Replicative ability wasassessed in RNA transfected Huh-7.5 cells by comparing the HCV RNAlevels to a SG-pol-control. Comparable levels of HCV RNA were observedat 96 h for replicons containing Ile or Val at position 2204, whereas anAla substitution resulted in a 3-fold reduction in HCV RNA compared toSG-5′HE (S2204I) (FIG. 5). In contrast, the remaining amino acidsubstitutions dramatically reduced HCV RNA to levels similar to theunmodified parental replicon, SG-5′HE (S2204) (˜1400-fold decrease; FIG.5). As expected, the relative HCV RNA levels were lower at 24 and 48 hafter transfection, however the levels were sufficient to assessreplicative ability at 48 h (FIG. 5). Although substitutions thatenhance subgenomic replication above that observed with S2204I were notfound, Val and Ala allowed efficient RNA replication.

The replication efficiency of subgenomic replicons carrying multipleadaptive mutations in NS5A was investigated. NS5A mutations S2197P,A2199T and S2204I independently enhance G418-resistant colony formationapproximately 2,500-, 15,000- and 20,000-fold, respectively (5). SG-5′HEreplicons (FIG. 1) carrying S2204I together with either A2199T, orA2199T and S2197P were constructed and HCV RNA levels in Huh-7.5 cellsmeasured by RT-PCR. Combining these NS5A mutations led to a reduction inHCV RNA levels compared to SG-5′HE (S2204I), with a 13-fold decrease forthe combination of A2199T and S2204I and negligible replication when allthree were combined (FIG. 6). Despite the observation that each of theseNSSA adaptive mutations alone enhanced replication, when combined, thereplicative ability of subgenomic RNAs declined, suggesting that thesecombinations are incompatible.

NS3 changes at positions 1112 (Q to R), 1202 (E to G) and 1280 (T to I)were engineered into SG-5′HE (S2204I) (FIG. 1) and their replicationcompared in Huh-7.5 cells by measuring HCV RNA levels, the frequency ofantigen-positive cells and by detection of ³⁵S-labeled proteins at 96 hfollowing transfection. Equivalent levels of HCV RNA relative to thepol⁻ RNA control were observed for each replicon (FIG. 7A). Thepercentage of NS5B-positive cells detected by FACS (˜30%; FIG. 7A) andimmunofluorescence (FIG. 7B) was also similar. However, the frequency ofNS3-positive cells was higher for replicons carrying the NS3 mutations(˜73-87%; FIG. 7A), which may simply reflect altered affinity of theNS3-specific antibody for these NS3 mutants. Finally, the levels ofimmunoprecipitated NS3, NS4B and NS5A were comparable (FIG. 7A).Although it was not verified that Q1112R alone was adaptive, Krieger andcoworkers previously reported that E1202G and T1280I alone or togetherincreased the replication efficiency by ˜13-, 6- and 25-fold,respectively (Krieger et al. 2001). These NS3 adaptive mutations do notfurther enhance replication when combined with S2204I in NS5A.

Example 13 Mutagenesis of the S2194 NS5A Phosphorylation Site

The role of NS5A phosphorylation in HCV replication remains a mystery.Previously, differences were noted in the extent of NS5A phosphorylationbetween replicons with different adaptive mutations in NS5A (Blight etal. 2000). For example, replicons with S2197C, S2197P or S2204Iexpressed minimal or no p58 as assessed by one-dimensional SDS-PAGEseparation of immunoprecipated NS5A, suggesting that NS5Ahyperphosphorylation is not essential for HCV replication. Recently,S2194 in NS5A of a subtype 1b isolate was identified as the-primary siteof p56 phosphorylation (Katze et al. 2000). To assess the possiblerequirement for phosphorylation of NS5A S2194, this residue was mutatedin SG-Neo (S2204I) (FIG. 1) to either Ala (S2194A+S2204I) or Asp(S2194D+S2204I), to ablate or mimic phosphorylation, respectively. G418transduction efficiencies of these replicons in Huh-7 cells wassignificantly lower than SG-Neo (S2204I) (120- and 17-fold lower; FIG.8A). To rule out the possibility that G418-resistant foci were generatedby reversion at this locus, the NS5A coding region was amplified fromtotal cellular RNA by RT-PCR and directly sequenced. The original Alaand Asp substitutions at position 2194 were confirmed (data not shown).To minimize the impact of possible second site compensating changes, HCVRNA and protein expression was measured 96 h after RNA transfection ofHuh-7.5 cells. The HCV RNA levels of S2194A+S2204I and S2194D+S2204Irelative to the pol control were ˜37- and 5-fold lower than SG-Neo(S2204I) (FIG. 8B), consistent with their reduced ability to renderHuh-7 cells G418 resistant. In addition, a lower frequency ofNS5B-positive cells was evident in S2194A+S2204I than in S2194D+S2204I(data not shown), and ³⁵S-labeled NS3 and NS4B were only detectable inHuh-7.5 cells transfected with SG-Neo (S2204I) and S2194D+S2204I (FIG.8B). It was not possible to directly study the phosphorylation status ofNS5A expressed from S2194A+S2204I and S2194D+S2204I since the levels ofNS5A expressed in transiently transfected cells were below the detectionlimit (FIG. 8B and data not shown). Although the quantitativedifferences in G418 transduction and replication efficiencies aredifficult to interpret given the possible incompatibility of combiningthe S2194 substitutions with the S2204I adaptive change, these data showthat phosphorylation of S2194 is not an absolute requirement for HCVreplication.

FIGURE LEGENDS

FIG. 1. Schematic representation of HCV RNAs used in this study. The 5′and 3′ NTR structures are shown and ORFs depicted as open boxes with thepolyprotein cleavage products indicated. The first 12 amino acids of thecore-coding region (solid box), the neo gene (Neo; shaded box), the EMCVIRES (EMCV; solid line) and ubiquitin (hatched box) are illustrated.Locations of the NS5A adaptive mutations S2204I (*) and Δ47aa areindicated.

FIG. 2. Identification of Huh-7 lines highly permissive for HCVreplication. Huh-7 cells that had been cured of self-replicatingsubgenomic RNAs by extended IFN treatment were electroporated with 1 μgof the subgenomic replicons, SG-Neo (S2204I), SG-Neo (5AΔ47) and SG-Neo(wt). Forty-eight hours later, cells were subjected to G418 selectionand the resulting colonies fixed and stained with crystal violet.Representative plates are illustrated with the number of transfectedcells seeded per 100-mm diameter dish shown on the left. Numbers beloweach dish refer to the calculated G418 transduction efficiency of thereplicon. To determine the G418 transduction efficiency, transfectedcells were serially titrated from 5×10⁵ to 10³ cells per 100-mm diameterdish, together with feeder cells electroporated with the pol⁻ replicon.The resulting G418-resistant foci were counted-for at least 3 celldensities and the relative G418 transduction efficiency expressed as apercentage, after dividing the number of colonies by the number ofelectroporated cells initially plated. Similar transduction efficiencieswere obtained in two independent transfections. A poliovirus subgenomicreplicon expressing GFP (see Methods) was electroporated in parallel.Based on both the fraction of GFP-positive cells and replicon-inducedcytopathogenicity, ˜90% of cells were routinely transfected. NT=nottested

FIG. 3. Detection of HCV proteins and RNA in Huh-7.5 and Huh-7 cellstransiently transfected with HCV RNA. Top panel, Huh-7.5 and Huh-7 cellswere transfected with the subgenomic replicons, pol⁻ (lanes 1 and 7),SG-5′HE (S2204I) (lanes 2 and 8), SG-5′HE (5AΔ47) (lanes 3 and 9),SG-Neo (S2204I) (lanes 4 and 10), SG-Neo (5AΔ47) (lanes 5 and 11) and FL(S2204I) HCV RNA (lanes 6 and 12). At 96 h posttransfection, monolayerswere incubated for 10 h in the presence of ³⁵S-methionine and -cysteine.Labeled cells were lysed, immunopreciptated with HCV-positive humanserum (JHF, anti-NS3, NS4B and NS5A) and labeled proteins separated bySDS-10% PAGE. Note that twice the amount of immunopreciptated sample wasloaded in lanes 6 and 12 (2×). The mobilities of molecular massstandards (MW) are indicated on the left and the migration of NS3, NS4B,NS5A and 5AΔ47 are shown on the right. Middle panel, Total cellular RNAwas extracted at 96 h posttransfection and quantified for HCV RNA levelsas described in the Materials and Methods. The ratio of HCV RNA relativeto the pol⁻ defective replicon is shown (HCV RNA/pol). HCV RNA levelsrelative to the por control were comparable in three independentexperiments. Lower two panels, 96 h after transfection cells were fixedwith 4% paraformaldehyde, permeabilized with 0.1% saponin, stained foreither HCV core or NS3 antigens and analyzed by FACS. The percentage ofcells expressing core and NS3 relative to an isotype matched irrelevantIgG is displayed. Values <1.5% were considered negative (−).

FIG. 4. HCV RNA accumulation after transfection of Huh-7.5 cells withfull-length HCV RNA. One-μg of in vitro transcribed RNA waselectroporated into Huh-7.5 and 2×10⁵ cells plated into 35-mm diameterwells. Total cellular RNA was isolated at 24, 48 and 96 hposttransfection and HCV RNA levels quantified as described in theMaterials and Methods. The ratio of HCV RNA relative to the pol⁻defective subgenomic RNA (HCV RNA/pol⁻) was plotted against the timeposttransfection and similar results were obtained when this experimentwas repeated

FIG. 5. Effects of alternative substitutions at position 2204 on HCV RNAreplication. Huh-7.5 cells were transfected with 1 μg of the SG-5′HEreplicons carrying the indicated amino acid substitutions and 2×10⁵cells plated in 35-mm diameter wells. After 24, 48 and 96 h in culture,total cellular RNA was extracted and HCV RNA levels measured asdescribed in the Materials and Methods. The ratio of HCV RNA relative tothe pol⁻ defective subgenomic RNA (HCV RNA/pol⁻) was plotted against thetime posttransfection. The increase in HCV RNA above pol⁻ is indicatedabove each bar. In this figure the levels of HCV RNA relative to thepol⁻ are the highest we have achieved so far. When these RNAs weretransfected into Huh-7.5 cells a second time a similar trend in HCV RNAaccumulation was observed.

FIG. 6. Effect(s) of combining NS5A adaptive mutations on HCV RNAreplication. Subgenomic replicons were transfected into Huh-7.5 cellsand HCV RNA levels quantitated as described in FIG. 5. The ratio of HCVRNA relative to the pol⁻ defective subgenomic RNA (HCV RNA/pol⁻) wasplotted against the time posttransfection and the increase in HCV RNAabove pol⁻ is indicated above each bar. An additional transfectionexperiment yielded HCV RNA/por ratios similar to those illustrated here.

FIG. 7. Effect(s) of combining NS3 and NS5A mutations on HCV RNAreplication. Subgenomic replicons lacking neo were generated carryingS2204I with further mutations in NS3. (A) Top, 96 h after RNAtransfection of Huh-7.5 cells, monolayers were labeled with ³⁵S-proteinlabeling mixture, lysed and NS3, NS4A and NS5A analyzed byimmunoprecipitation, SDS-10% PAGE and autoradiography. Positions of themolecular weight standards are given on the left and HCV-specificproteins indicated to the right. Middle, Total cellular RNA wasextracted at 96 h posttransfection and HCV RNA levels quantified asdescribed in the Materials and Methods. The ratio of HCV RNA relative tothe pol⁻ negative control is shown (HCV RNA/pol⁻). Comparable ratioswere obtained in two independent experiments. Lower two panels, 96 hafter transfection, cells were fixed with 4% paraformaldehyde,permeabilized with 0.1% saponin, stained for HCV NS3 and NS5B antigensand analyzed by FACS. The percentage of cells expressing NS3 and NS5Brelative to an isotype matched irrelevant IgG is displayed. Values <1.5%were considered negative (−). (B) Transfected cells seeded in eight-wellchamber slides were fixed, permeablized and stained for NS5B byimmnunofluorescence after 96 h in culture. Nuclei were counterstainedwith Hoescht 33342 and stained cells visualized by fluorescentmicroscopy (×40 magnification).

FIG. 8. Effect of S2194A and S2194D mutations on HCV RNA replication.S2194 was replaced with Ala or Asp in the selectable bicistronicreplicon SG-Neo (S2204I) and RNA transcribed in vitro. (A) RNAtranscripts were transfected into Huh-7 cells and G418-selected coloniesfixed and stained with crystal violet. The relative G418 transductionefficiencies are indicated below each dish. (B) Ninety-six hoursposttransfection Huh-7.5 cells were labeled with ³⁵S-methionine and-cysteine for 10 h. Cells were lysed, and HCV proteins isolated byimmunoprecipitation using a patient serum specific for NS3, NS4B andNS5A. HCV proteins and the positions of protein molecular weightstandards (in kilodaltons) are shown. The ratio of HCV RNA relative tothe pol⁻ negative control at 96 h posttransfection is shown below eachtrack (HCV RNA/pol⁻). The results illustrated are representative of twoindependent transfections.

As various modifications could be made in the constructions and methodsherein described and illustrated without departing from the scope of theinvention, it is intended that all matter contained in the foregoingdescription or shown in the accompanying drawings shall be interpretedas illustrative rather than limiting. Thus, the breadth and scope of thepresent invention should not be limited by any of the above-describedexemplary embodiments. TABLE 1 Oligodeoxynucleotides used in this study.Name Sequence  885 (−)CCCTCTAGAACGCCCCGAAACCTAGGGTGGCG 1030 (−)CCCTCTAGACTCGAG GGAATTTCCTGGAC 1184 (+)GACG GCTAAGCGTAGGCTGGCCAGGGGATCTCCCCCCTCCTTGGCCAGCTCATCAGCT GAC CAGCTGTCTGCGCCTTCC1287 (+)AGACC GTGCAC CAGACCACAACGGTTTCCCTCTAGCGGGATCAATTCCG 1288(−)CCAGT AACGTT AGGGGGGGGGGAGGGAGAGGGGCGGAATTGATCCCGCT 1289 (+)CCAAAGGGCGCGCC ATGCAGATCTTCGTGAAGACC 3′ 1290 (−)AATAG GAGCTC CACCGCGGAGACGC1291 (+)CGGTG GAGCTC CTATTACGGCCTACTCCCAAC 1292 (−)ATTGG TGTACATTTGGGTGATTGG 1293 (+)TCTGG AAGCTT CTTGAAGACA 1294 (−)GGCTT GACGTCCTGTGGGCGGCGGTTGGTGTTACGTTTGGTTTTTCTTTGAGGTTTAGGATTCGTGCTCATTATTATCGTGTTTTTCAAAGG1319 (+)AGACG GCTAAGC GTAGGCTGGCCAGGGGATCTCCCCCCTCCTTGGCCAGCTCATCAGCTGTA CAGCTGTCTGCGCCTTCC 1320 (+)AGACG GCTAAGCGTAGGCTGGCCAGGGGATCTCCCCCCTCCTTGGCCAGCTCATCAGCT GCC CAGCTGTCTGCGCCTTCC1322 (+)AGACG GCTAAGC GTAGGCTGGCCAGGGGATCTCCCCCCTCCTTGGCCAGCTCATCAGCTTAC CAGCTGTCTGCGCCTTCC 1324 (+)AGACG GCTAAGCGTAGGCTGGCCAGGGGATCTCCCCCCTCCTTGGCCAGCTCATCAGCT GAA CAGCTGTCTGCGCCTTCC1325 (+)AGACG GCTAAGC GTAGGCTGGCCAGGGGATCTCCCCCCTCCTTGGCCAGCTCATCAGCTACA CAGCTGTCTGCGCCTTCC 1326 (+)AGACG GCTAAGCGTAGGCTGGCCAGGGGATCTCCCCCCTCCTTG ACC AGCTCATCAGCTATCCAGCTGTCTGCGCCTTCC1327 (+)AGACG GCTAAGC GTAGGCTGGCCAGGGGATCTCCCCCC CCC TTG ACCAGCTCATCAGCTATCCAGCTGTCTGCGCCTTCC 1356 (−)CCGC TCTAGATACGTGATGGGGGCACCCGTGGTGATGGTCCTTACCCC GAT TCTGATGTTAGGGTCGATAC 1358(+)CCGA TGTACA CCAATGTGGACCAGGACCTCGTCGGCTGG CGA GCGCCCCCCGGGGCGCGTTCC1359 (+)CCGC GTGCAC CCGAGGGGTTGCGAAGGCGGTGGACTTTGTACCCGTCGAGTCTATG GGAACCACTATGCGGTCCCCGGTC 5′Ala (+)CCAC GCTAAGC GTAGGCTGGCCAGGGGA GCACCCCCCTCCTTGGCCAGCTC 5′Asp (+)CCAC GCTAAGC GTAGGCTGGCCAGGGGA GATCCCCCCTCCTTGGCCAGCTC^(a)Nucleotide changes are highlighted in bold and the resultant codonis underlined^(b)Restriction sites used for cDNA cloning are underlined^(c)The polarities of oligonucleotides are indicated either the HCVgenome RNA sense (+) or its complement (−)

1. A method for producing a cell line permissive for hepatitis C virus(HCV) replication comprising (a) culturing cells infected with HCV; (b)curing said cells of (a) of HCV; and (c) identifying a subline of saidcured cells of (b) that is permissive for HCV replication.
 2. The methodof claim 1, wherein said curing step of (b) comprises subjecting saidinfected cells of (a) to treatment with an antiviral agent.
 3. Themethod of claim 2, wherein said antiviral agent is an antiviralcytokine.
 4. The method of claim 3, wherein said antiviral cytokine isinterferon.
 5. The method of claim 1, wherein said cells of (a) arevertebrate cells.
 6. The method of claim 5, wherein said vertebratecells are human cells.
 7. The method of claim 1, wherein said cells of(a) are hepatocyte cells.
 8. The method of claim 7, wherein saidhepatocyte cells are human hepatocyte cells.
 9. The method of claim 1wherein said subline of (c) supports HCV replication at a frequency ofat least 30%.
 10. A method for producing a cell line permissive for HCVreplication, the method comprising (a) providing a cell line thatcomprises a replicating genomic or subgenomic HCV RNA (b) curing saidcell line of (a) of HCV RNA (c) identifying sublines of said cured cellline of (b) that are permissive for HCV replication.
 11. The method ofclaim 10, wherein said curing of step (b) comprises treatment with anantiviral agent.
 12. The method of claim 11, wherein said agent is anantiviral cytokine.
 13. The method of claim 12, wherein said antiviralcytokine is interferon.
 14. The method of claim 10 wherein said cellline of (a) comprises a replicating subgenomic HCV RNA containing noadaptive mutations.
 15. The method of claim 10, wherein said cell lineof (a) comprises a replicating subgenomic HCV RNA that comprises anadaptive mutation.
 16. A cell line that is permissive for HCVreplication, wherein said cell line is produced by curing a host cellline infected with HCV and then selecting cured sublines that arepermissive for HCV replication.
 17. A cell line according to claim 16,wherein said curing comprises treating said host cell line withinterferon.
 18. A cell line that is permissive for HCV RNA replication,wherein said cell line has been cured of HCV RNA by treatment withinterferon.
 19. A method for producing a cell line that is permissivefor HCV RNA replication, the method comprising (a) transfecting hostcells with replicating HCV RNA (b) subjecting said host cells toconditions that cure said host cells of HCV RNA (c) selecting cured cellpopulations of (b) (d) growing the selected cured cell populations of(c) to generate a cell line that is permissive for HCV RNA replication.20. The method according to claim 19, wherein said HCV RNA of step (a)is subgenomic HCV RNA.
 21. The method according to claim 19, whereinstep (b) comprises treating said host cells with an antiviral agent. 22.The method of claim 21 wherein said antiviral agent is an antiviralcytokine.
 23. The method according to claim 21, wherein said antiviralcytokine is interferon.
 24. The method of claim 21, wherein saidinterferon is interferon-α.
 25. The method according to claim 19,wherein said cell line of (d) supports HCV RNA replication at afrequency of between about 10% and about 75%.
 26. The method accordingto claim 25, wherein said cell line of (d) supports HCV RNA replicationat a frequency of between about 10% and about 30%.
 27. The methodaccording to claim 25, wherein said cell line of (d) supports HCV RNAreplication at a frequency of at least 30%.
 28. The method according toclaim 27, wherein said cell line of (d) supports HCV RNA replication ata frequency of at least 50%.
 29. The method according to claim 19,wherein said host cell is a vertebrate cell.
 30. The method according toclaim 29, wherein said host cell is a human cell.
 31. The methodaccording to claim 29, wherein said host cell is a hepatocyte cell. 32.The method according to claim 30, wherein said host cell is a humancell.
 33. A cell line produced-by the method of claim
 19. 34. A cellline according to claim 33, wherein said host cell contains subgenomicHCV RNA.
 35. A cell line according to claim 34, wherein said subgenomicHCV RNA comprises an adaptive mutation.
 36. The cell line of claim 35,wherein said adaptive mutation is S2204I, said position being identifiedby alignment with the genotype 1b Con1 full-length HCV genome (GenbankAccession no. AJ238799) commencing with the core-coding region.
 37. Acell line produced by the method of claim
 1. 38. A cell line produced bythe method of claim 10.